Osteosarcoma (OS) is an aggressive malignant primary bone cancer with a high propensity for lung metastasis. OS frequently originates in the long bones during periods of rapid bone growth. Consequently, OS represents the most prevalent bone cancers affecting children and adolescent and young adults (AYA), with ~400-600 cases a year and accounts roughly half of all new cases of OS diagnosed in the United States. Despite aggressive combination chemotherapy and surgery, the outcome for metastatic OS remains dismal, and the overall survival in children and AYA patients with metastatic OS has not improved significantly over the past 3 decades. A high proportion of OS patients develop pulmonary metastasis (pOS) either at the time of diagnosis (20%) or after initiation of multimodal therapy including combination chemotherapy and surgery. Unfortunately, almost all of the patients who develop surgically un-resectable pOS invariably succumb to this devastating disease. Therefore, pOS represent a disease with unmet needs. As OS contains extremely complex genetic alterations, molecular targeted precision therapy has proven challenging. Recent exciting scientific development implicates the immune system as a potential important new armamentarium as a novel approach to control or reduce pOS. In the current proposal, we identified tumor-expressed Vascular Cell Adhesion Molecule-1 (tVCAM-1) as playing a pivotal role in immune-mediated cancer metastasis by interacting with ?4?1 integrin on lung macrophages (MACs), thereby implicating the pulmonary macrophages as a major culprit in the pathophysiology of metastatic OS. Compared to non-metastatic parental tumor (K7), metastatic murine OS cells (K7M2) express high surface VCAM-1, and absence of either tVCAM-1 or MACs prevents the development and ameliorates a large proportion of established pOS. Therefore, we wish to further examine our proposal that a targetable cellular and molecular mechanism, VCAM-1/ ?4?1, is a primary driver of OS-MAC interaction, providing the suitable lung tissue niche for pOS in vivo. Our hypothesis is that interfering VCAM- 1/?4?1 signaling between pOS and MACs by down-regulating VCAM-1, depleting MACs or disrupting VCAM- 1-?4?1 signaling will reduce pulmonary metastasis and improve survival. Specific Aim 1: We will characterize functional outcomes of MACS and other immune cells following VCAM-1/?4?4 signaling engagement with pOS. We will compare VCAM-1lo and VCAM-1hi pOS cells for in vivo growth and associated immune responses in the lungs, and expand our observation to include testing lung metastatic potential of other human and mouse pOS cell lines and PDXs available through our own PDX repository and that of our collaborator at Texas Children's Hospital. We will characterize phenotypic and functional outcomes of various myeloid and other immune cellular compartment in the lung tissue of VCAM-1lo and VCAM-1hi pOS cells using cellular, molecular and imaging approaches. Specific Aim 2: We will test the efficacy of functional blockade of VCAM- 1/?4?1 interaction by depleting MACs with intranasal liposomal clodronate treatment or by inhibiting molecular interactions using VCAM-1 specific inhibitory peptide (iVCAM-1p) or intranasal / intratracheal administration of anti-a4 blocking antibody. In the latter scenario involving anti-?4 blocking antibody, we set a goal to obtain foundational pre-clinical, IND-enabling data for monitoring systemic and local toxicities and refining optimal dosing in order to rapidly translate findings from this current grant into Phase I studies using the FDA-approved anti-?4 antibody, Natalizumab, via the intratracheal and inhalation routes for the treatment of patients with late- stage pOS, for whom no other treatment options with demonstrable therapeutic benefits are available. Success in this high priority effort could have a profound effect in improving the outcome and quality of life for children and AYA patients afflicted with this devastating disease.